This proposal outlines a strategy for the design, synthesis, and evaluation of a new class of inhibitors for serine and cysteine proteases. These inhibitors are based upon a 4-heterocyclohexanone ring, and are designed to react with the enzyme active site nucleophile to give a reversibly formed hemithioketal. The electrophilicity of the ketone in these inhibitors is enhanced by ring strain and by through- space electrostatic repulsion with the heteroatom at the 1-position of the ring. Equilibrium constants for the addition of water and 3 mercaptoproprionic acid to several simple 4-heterocyclohexanones are measured by proton NMR spectroscopy. These reactions are models for the addition of the enzyme active site nucleophile to the corresponding inhibitors. The equilibrium constants give a linear correlation with the field substituent constant F for the functional group at the 1-position of the heterocyclohexanone. These results demonstrate that the combination of ring strain and through-space electrostatic repulsion can stabilize the hemithioketal of a 4-heterocyclohexanone by more than 5.5 kcal/mol compared to a hemithioketal of an unactivated ketone such as acetone. In order to prove the validity of this inhibitor design, a series of 4- heterocyclohexanone-based inhibitors of the prototypical cysteine protease papain have been synthesized. Inhibition constants, which range from 11 to 120 micromolar, are found to give a linear correlation with the equilibrium constants for addition of 3-mercaptoproprionic acid to simple 4-heterocyclohexanones. Thus the model system is a good method for predicting the potency of structurally related enzyme inhibitors. This work will be extended in three specific areas. First, the mechanism of protease inhibition will be investigated by synthesis of a tetrahydropyranone inhibitor which as a carbon-13 label incorporated at the ketone position. The covalent hemithioketal adduct between the inhibitor and a stoichiometric amount of enzyme will be observed using 13C NMR. Second, inhibition of the serine protease plasmin will be investigated. These studies will confirm that 4-heterocyclohexanone- based inhibitors are active against both cysteine and serine proteases. In addition, they will provide new inhibitors for an enzyme that is implicated in metastasis of cancer. Finally, the potency and specificity of these inhibitors will be increases by extending their interactions into the leaving group subsites of the enzyme active site. This will be accomplished by substituting both the 3 and 5 positions of the 4-heterocyclohexanone ring with appropriate functionality. The strategy will be applied to the synthesis of inhibitors for cathepsin B.